Use of quinoline derivatives with anti-integrase effect and applications thereof

ABSTRACT

The invention relates to the use of 8-hydroxyquinoline 7-carboxylic acid derivatives in order to produce integrase-inhibiting medicaments, capable of blocking viral replication in the stages preceding integration, and if appropriate at the level of this integration stage, these medicaments being usable for the treatment of retroviral pathologies, in particular for the treatment of AIDS.

A subject of the invention is the use of quinoline derivatives withanti-integrase effect and its applications.

The inhibiting effect of quinoline derivatives at the level of theintegration of viral DNA into a host cell has already been reported.

In particular, such derivatives are described in Application FR 97 04289 of 8th Apr. 1997 and Application FR 01 13209 of 12th Oct. 2001.

Study of these derivatives has shown that they act as competitors ofviral DNA for binding to integrase.

The work of the inventors has now made it possible to demonstrateeffects of the latter on the stages preceding the integration of viralDNA, in particular on reverse transcription and nuclear translocation.

The invention therefore has the purpose of providing a novel use ofthese quinoline derivatives in order to produce medicaments with aninhibiting effect on the activity of integrase, covering differentstages after the entry of the virus into the cell, and in particularreverse transcription, nuclear translocation of viral DNA and integrase,and if appropriate the integration of viral DNA into the genome of thehost cell.

The invention relates to the use of 8-hydroxyquinoline 7-carboxylic acidderivatives, or its pharmaceutically acceptable salts, in order toproduce integrase-inhibiting medicaments, capable of blocking viralreplication in the stages preceding integration, and if appropriate atthe level of this integration stage, these medicaments being usable forthe treatment of retroviral pathologies, in particular for the treatmentof AIDS.

The derivatives used according to the invention are more particularlycompetitors of viral DNA for binding to integrase.

According to an embodiment of the invention, these are styrylquinolines,advantageously those defined in Application FR 97 04 289. It is recalledthat, according to the most general definition given in thisApplication, these derivatives correspond to formula I

in which

-   -   R_(b) and R_(c), identical to or different from one another,        represent one or more substituents, themselves identical or        different, occupying any position on the rings, this substituent        or these substituents being chosen from a —(CH₂), —Y or —CH═CH—Y        group, where Y represents a halogen atom, an —OH, —OR, —COH,        —COR, —COOH, —COOR, —COH, —COR, —CONH₂, —CON (R_(x), R_(y))        —CH═NOH, —CO—CH═NOH, —NH₂, —N(R_(x), R_(y)), —NO₂, —PO(OR)₂—SH₂,        —SR, —SO₂R, —SO₂NHR, CN, or Z(R_(c)) radical, where R represents        an alkyl radical with 1 to 8 carbon atoms, or an aryl or        heterocyclic radical, R_(x) and R_(y), identical or different        represent an alkyl radical with 1 to 5 carbon atoms, Z        represents an aryl or heterocyclic radical and n is zero or an        integer from 1 to 5,    -   R_(b) moreover being able to represent a hydrogen atom,    -   and when Y represents a —COOH or —COOR group in R_(c), Z, if it        represents an aryl group, comprises at least 3 substituents or        the quinoline nucleus is tri-substituted,    -   —X represents an ethylenic double bond, a —(CH₂)_(n)— group,        where n is an integer from 1 to 5, or a —CH (R_(d))—CH(R_(e))—        group, R_(d) and R_(e), identical or different, representing a        hydrogen, halogen atom, a hydroxy or epoxy group,    -   as well as the pharmaceutically acceptable salts of these        derivatives, their diastereoisomeric forms and their        enantiomeric forms.

The invention quite particularly relates to the use of8-hydroxy-2-[2-[(3,4-dihydroxy, 5-methoxyphenyl) ethenyl]]7-quinolinecarboxylic acid, hereafter designated FZ41, of formula II

According to another embodiment of the invention, the derivatives usedin order to produce said medicaments are 2-carbamoyl-8-hydroxyquinoline7-carboxylic acid derivatives. In particular the invention relates tothe use of the derivatives according to Patent Application FR 01 13 209.These derivatives, in their most general definition, are characterizedin that they correspond to formula III

in which

-   -   —X′ represents a —(CH₂)_(n)— alkyl chain in which n is equal to        0, 1 or 2, O, or N,    -   —Z′ represents an aromatic ring which can comprise heteroatoms        chosen from O, N or S, in substitution of the carbon atoms        constituting said aromatic ring, this ring being able to be        substituted or not substituted by Rb′,    -   —Rb′ represents 1 to 3 identical or different substituents,        chosen from the —OH, —OR, —COOH, —COOR, —COH, —COR, —NH₂,        —NH(R), —NH(R,R′), —SH and —SR and CN groups,    -   Ra′ represents a hydrogen atom or a —(CH₂)_(n)—Y′ group, for        which n′ is equal to 0, 1, 2 or 3 and Y′ represents —CH₃, —COOH,        —COOR, —CN, —OH, —OR, SR, or an aryl group optionally        substituted by Rb′,    -   —R and R′, identical or different, represent a linear or        branched alkyl chain with 1 to 4 carbon atoms, and their        pharmaceutically acceptable salts.

Study of the properties of the derivatives used according to theinvention has made it possible to demonstrate their effect on the stagesprior to the integration of the viral DNA into the genome of the hostcell with, if appropriate, an inhibiting effect at the level of theintegration stage. These effects are directed against the reversetranscription of the retrovirus RNA of animal and human origin, and inparticular of HIV-1, HIV-2, SIV, RSV and against the nucleartranslocation of these retroviruses. The effectiveness of thesederivatives is observed at submicromolar concentrations. IC₅₀s notexceeding 1 μM, advantageously 0.5 μM

-   -   and even 0.1 μM are thus obtained.

These derivatives used according to the invention moreover have theadvantage of considerable harmlessness and satisfactory bioavailibilityof the active drug.

These properties are therefore turned to good account according to theinvention by using said derivatives as active ingredients of medicamentsin combination with pharmaceutically acceptable vehicles. They can alsobe advantageously used in combination for multitherapies.

The dosages and administration methods are adapted depending on thetreatment for each mono- or multitherapy used.

For multitherapies, said derivatives are advantageously presented in thesame packaging in the form of kits.

Other characteristics and advantages of the invention are described inthe examples which follow. In these examples, reference is made to FIGS.1 and 2, which represent, respectively:

FIG. 1, the quantification of the total viral DNA synthesized 6 hoursafter infection and the quantity of viral DNA integrated into the genome8 hours after infection, and

FIG. 2, the quantity of RNA and that of genomic DNA having entered 1hour 30 minutes after infection, and the total DNA synthesized 6 hoursafter infection.

Study of the Quantification of the Total Viral DNA Synthesized and ofthe DNA Integrated into the Genome of a Host Cell after Infection

The operation described by Butler et al. in Nat. Med. 2001 May; 7 (5):631-4 is carried out.

As shown in FIG. 1, it is noted with the molecule L731-988 (published byHazuda et al. in Science, 28th Jan. 2000, 287 (5453): 646-50) that theDNA is perfectly synthesized, but only slightly or not at all integratedinto the genome of the cell.

On the other hand, when administrating the FZ41, a strong reduction inthe synthesis of viral DNA and in integration is noted.

Determination of the Quantity of RNA and Genomic DNA After Infection andof Total DNA Synthesized

As control a non-nucleoside RT inhibitor, namely nevirapine (NNRTI) wasused and, as products to be tested, L731-988 or FZ41.

The treatments carried out with these products do not lead to areduction in the genomic RNA entering the cell 1 hour 30 minutes afterinfection. When comparing the data obtained at the start and at the endof the RT stage, the strong effect of the product used according to theinvention and according to the control is noted.

Study of the Effect on the Viral Replication Cycle

FIG. 3 shows the quantifications of the viral nucleic species byquantitative PCR in cells infected by HIV and treated or not treatedwith FZ41, a non-specific inhibitor of the entry of the virus (dextransulphate) or an integration inhibitor (L731-988).

A: quantification by Q-PCR of the intracellular genomic RNA.

B: quantification by Q-PCR of the cDNA retrotranscribed at the start ofretrotranscription

C: quantification by Q-PCR of the cDNA retrotranscribed at the end ofretrotranscription

D: quantification by Q-PCR of the cDNA integrated into the host genome

The results obtained show that styrylquinoline derivatives having aninhibiting activity on integrase and more particularly containing thepharmacophore described in patents FR 2 761 687 A and FR 01 13 209inhibit the synthesis of retroviral DNA from the start of reversetranscription, but do not affect the quantity of viral genomic RNApenetrating into the cells.

Selection of Resistant Mutants

Culture of the virus in the presence of increasing concentrations ofFZ41 allowed the emergence of viruses resistant to the drug. Themutations which appeared in these viruses are situated in the integraseat positions V165 and V249 on the one hand and at position C280 for asecond virus. The mutants were generated by site specific mutagenesisand their resistance was verified.

FIG. 4 shows the resistance index obtained for each of the viruses,compare with the wild-type virus.

Effect of styrylguinolines on the Nuclear Translocation of the Integrase

FIGS. 5 and 6 show the quantification of the fluorescence intensitymeasured in a nuclear import test on the integrase of HIV-1 inpermeabilized HeLa cells in the presence or absence of cytoplasmicextracts.

The import tests in FIG. 5 were carried out as described in Depienne etal. 2001. HeLa cells were permeabilized with Digitonin, then incubatedfor 30 minutes at 30° C. with integrase coupled with Cyanine 3fluorochrome in the presence of energy, in the absence of cytosolicextracts and in the presence or absence of increasing concentrations ofdifferent molecules as indicated. The cells were then fixed, analyzed byepifluorescence microscopy and acquisitions were carried out with a CCDcamera.

For each condition, the fluorescence intensity per surface unit wasquantified in 150 to 300 nuclei originating from 3 independentexperiments.

FIG. 7 shows the quantification of the fluorescence intensity measuredin a BSA-NLS nuclear import test in permeabilized HeLa cells

The formulae of molecules used are given in the table below. MoleculeFormula Integrase inhibitor in vitro FZ41

YES KHD161

YES BioA53

YES FZ117

NO L731-988

YES

The results obtained show that the styrylquinoline derivatives having aninhibiting activity on integrase and more particularly containing thepharmacophore described in patents FR 2 761 687 A and FR 01 13 209inhibit the nuclear translocation of HIV integrase.

Thus, the FZ41 and KHD161 molecules considerably reduce the quantity offluorescence obtained in the nuclei of permeabilized cells aftertreatment.

A contrario, the addition to the experiment of other integraseinhibitors (L731-988) or inactive styrylquinoline derivatives (FZ117)does not modify the nuclear import of the integrase.

The inhibition of this integrase translocation is specific since thenuclear import of BSA-NLS (Bovine serum albumin conjugated to a nuclearimport signal) is not affected by the drugs (FIG. 6).

Moreover, the mutations obtained by selection with the FZ41 molecule andmore particularly the V1651/V2491 mutations lead to a lack of thenuclear import of integrase in experiments on protein expression ineucaryotic cells.

The data of FIGS. 6 and 7 were obtained as follows: HeLa cells werepermeabilized with digitonin, then incubated for 30 minutes at 30° C.with integrase conjugated to Cyanine 3 fluorochrome and with bovineserum albumin (BSA) conjugated to the standard nuclear localizationsignal of the T antigen of the SV40 virus (NLS) and with fluorescein(BSA-NLS), in the presence of cytosolic extracts and energy and in thepresence or in the absence of increasing concentrations of differentmolecules as indicated. The cells were then fixed, analyzed byepifluorescence microscopy and acquisitions were carried out with a CCDcamera. For each condition, the fluorescence intensity per surface unitwas quantified in 150 to 300 nuclei originating from 3 independentexperiments. FIG. 6 represents the results obtained with integrase andFIG. 7 those obtained with BSA-NLS.

BIBLIOGRAPHICAL REFERENCE

-   Depienne C, Mousnier A, Leh H, Le Rouzic E, Dormont D, Benichou S,    Dargemont C. Characterization of the nuclear import pathway for    HIV-1 integrase. J Biol Chem. May 25, 2001;276(21): 18102-7.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. Method for inhibitingintegrase and blocking viral replication in the stages precedingintegration, and if appropriate at the level of this integration stage,for the treatment of retroviral pathologies, comprising administering a8-hydroxyquinoline 7-carboxylic acid derivative.
 10. Method according toclaim 9, for the treatment of AIDS.
 11. Method according to claim 9 or10, wherein said derivatives are styrylquinolines.
 12. Method accordingto claim 11, wherein the styrylquinolines have formula l:

wherein Rb and Rc, identical to or different from one another, representone or more substituents, themselves identical or different, occupyingany position on the rings, this substituent or these substituents beingchosen from a —(CH₂)_(n)— Y or —CH═CH—Y group, where Y represents ahalogen atom, an —OH, —OR, —COH, —COR, —COOH, —COOR, —CONH₂, —CON(R_(x),R_(y)) —CH═NOH, —CO—CH═NOH, —NH₂, —N(Rx, Ry), —NO₂, —PO(OR)₂, —SH₂, —SR,—SO₂R, —SO₂NHR or CN, where R represents an alkyl radical with 1 to 8carbon atoms, aryl or heterocyclic radical, Rx and Ry, identical ordifferent, represent an alkyl radical with 1 to 5 carbon atoms, Zrepresents an aryl or heterocyclic radical and n is zero or an integerfrom 1 to 5, Rb moreover being able to represent a hydrogen atom, andwhen Y represents a —COOH or —COOR group in Rc, Z, if it represents anaryl group, comprises at least 3 substituents or the quinoline nucleusis tri-substituted, X represents an ethylenic double bond, or a—(CH₂)_(n)— group, where n is an integer from 1 to 5, or a—CH(Rd)-CH(Re)- group, Rd and Re, identical or different, representing ahydrogen, halogen atom, a hydroxy or epoxy group, as well as thepharmaceutically acceptable salts of these derivatives, theirdiastereoisomeric forms and their enantiomeric forms.
 13. Methodaccording to claim 1 2, characterized in that said derivative is8-hydroyx-2-[2-[3,4-dihydroxy, 5-methoxyphenyl)ethenyl]]7-quinolinecarboxylic acid.
 14. Method according to claim 9 or 10, wherein saidderivatives are 2-carbamoyl-8-hydroxyquinoline 7-carboxylic acidderivatives.
 15. Method according to claim 14, wherein said derivativeshave formula III

wherein —X′ represents a —(CH₂)_(n)— alkyl chain in which n is equal to0, 1 or 2, 0, or N, —Z′ represents an aromatic ring which can compriseheteroatoms chosen from O, N or S, in substitution of the carbon atomsconstituting said aromatic ring, this ring being able to be substitutedor not substituted by Rb′, Rb′ represents 1 to 3 identical or differentsubstituents, chosen from the —OH, —OR, —COOH, —COOR, —COH, —COR, —NH₂,—NH(R), —NH(R,R′), —SH and —SR and CN groups, Ra′ represents a hydrogenatom or a —(CH₂)_(n)′—Y′ group, for which n′ is equal to 0, 1, 2 or 3and Y′ represents —CH₃, —COOH, —COOR, —CN, —OH, —OR, SR, or an arylgroup optionally substituted by Rb′, —R and R′, identical or different,represent a linear or branched alkyl chain with 1 to 4 carbon atoms, andtheir pharmaceutically acceptable salts.
 16. Method according to claim 9or 10, wherein said derivatives are administered in multitherapies. 17.Method for inhibiting integrase nuclear import, comprising administeringa derivative according to claim 12, 13 or 15.